Opioid-induced glial activation, which compromises pain treatment and contributes to the development of drug addiction and abuse, is regulated via a signaling pathway downstream of toll-like receptor-4 (TLR4), a membrane spanning receptor that functions in complex with its accessory protein MD-2. As current opioid pharmacotherapeutics have failed to control pain while avoiding the negative consequences, there is an urgent need to understand opioid dysregulation via TLR4. The central hypothesis of the current proposal is that disruption of the TLR-4/MD-2 complex formation can inhibit opioid-induced glial activation, thereby enhancing analgesia and reducing opioid tolerance and dependence. The rationale underlying the proposed research is that the identified inhibitors, which selectively block the critical protein-protein interactions between TLR4 and MD-2, will provide a useful tool for investigating the role of the TLR4-mediated signaling pathway in glial activation. The proposed research is innovative because it is the first drug discovery approach attempting to regulate opioid-induced glial activation. The proposed high risk/high reward approach, if successful, is projected to yield significant novel outcomes. First, the results will shed light on the mechanism of the clinically relevant opioid-induced glial activation. Second, if successful, the peptide and peptidomimetic antagonists of the TLR4/MD-2 interactions identified in the proposed research can serve as prototypes for more drug-like small-molecule inhibitors. These inhibitors may eventually find application in the development of novel therapeutics to enhance the clinical efficacy of opioid analgesics and to treat opioid addiction and abuse, as well as other clinically relevant indications. The proposed studies are built on a strong collaborative team with expertise that optimizes its chance to effectively bridge between atomic detail of the TLR4/MD-2 interaction and its macroscopic effect, namely pain management and avoiding negative consequences of opoid use. In Aim 1, antagonists of TLR4 or MD-2 that block the TLR4/MD-2 complex formation will be developed using a cutting-edge computational technology. The working hypothesis here is that conformationally strained peptides derived from the binding region can compete with the full-length protein and thereby inhibit the TLR4/MD-2 interaction. These peptides can serve as starting points for the computational design of stronger inhibitors. In Aim 2, the second working hypothesis, that the inhibitors of the TLR4/MD-2 interactions can non-competitively prevent opioids from inducing TLR4-mediated glial activation, will be tested. Cellular assays and animal models will be used to evaluate the inhibition of glial activation by the TLR4 antagonists both in vitro and in vivo. The proposed research is significant because it is expected to establish the TLR4/MD-2 protein-protein complex as a novel therapeutic target for optimizing opioid analgesia while preventing and treating opioid abuse. Regarding its positive impact on scientific advancements, this work will (1) improve scientific understanding of drug dependence and pain suppression and (2) allow the development of a new generation of therapeutics. PUBLIC HEALTH RELEVANCE: The proposed research aims to unravel the mechanism of opioid-induced glial activation that both hinders the ability of opioids to effectively control pain and also importantly contributes to the development of drug addiction and abuse. State-of-the-art technologies will be employed to define, design, create, and test new chemical entities predicted to prevent opioid induced glial activation, thereby optimizing opioid analgesia while preventing negative consequences of clinical opioid use.